Hepatocellular carcinoma (HCC) is one the most common forms of cancer worldwide, with approximately 1,000,000 new cases and 1,300,000 deaths reported annually. It is one of the most lethal forms of cancer, in part due to the fact that HCC cells are highly resistant to conventional cancer therapies. New approaches to therapy are desperately needed, and is the focus of the present application. Immunotoxins composed of a tumor specific targeting moiety, such as an antibody, attached to a cytotoxic molecule have shown some promise for cancer therapy. However, major drawbacks include poor penetration into solid tumors and the immunogenicity of non-human toxic moieties. This proposal will develop an HCC-specific immunotoxin that overcomes these problems. A single chain variable region antibody fragment (scFv) derived from a HCC-specific hybridoma will be genetically fused to a potently toxic 9 kD novel human peptide called "activator of DNA fragmentation" (ADF). Introduction of recombinant ADF at low nanomolar concentrations into cells is toxic to a wide variety of tumor cell types, including HCC's. Furthermore, tumor cell variants that are highly resistant to chemotherapeutic drugs through different mechanisms, are still just as sensitive to ADF as non-resistant tumor cells. The first aim of this application is to clone and express the anti-HCC scFv alone and fused to ADF (designated TH101). Cell binding and cytotoxicity studies will be performed to evalutate the specificity of the new construct on a variety of tumor cell lines as well as normal cell types. Initial studies will be performed to evaluate the potential acute toxicity of TH101 in mice, as well as the preferential accumulation of the conjugate in hepatoma xenografts as opposed to normal tissue. Phase II studies will test TH101 in mouse xenograft therapeutic models of HCC and develop a humanized version of the scFv construct for further clinical development. The successful completion of this project will result in a small completely human HCC-specific immunotoxin that will have significant advantages compared to immunotoxins prepared with bulky whole antibodies or immunogenic non-human toxin moieties. Furthermore, since ADF is so potently toxic to a wide variety of drug-resistant tumor types, it may also have wide application as a "bullet" to arm antibodies specific for different tumor cell types in future applications.